Modeling of the enzymatic kinetic synthesis of cephalexin - Influence of substrate concentration and temperature

During enzymatic kinetic synthesis of cephalexin, an activated phenylglycin e derivative (phenylglycine amide or phenylglycine methyl ester) is coupled to the nucleus 7-aminodeacetoxycephalosporanic acid (7-ADCA). Simultaneous ly, hydrolysis of phenylglycine amide and hydrolysis of cephalexin take pla ce. This results in a temporary high-product concentration that is subseque ntly consumed by the enzyme. To optimize productivity, it is necessary to d evelop models that predict the course of the reaction. Such models are know n from literature but these are only applicable for a limited range of expe rimental conditions. In this article a model is presented that is valid for a wide range of subs trate concentrations (0-490 mM for phenylglycine amide and 0-300 mM for 7-A DCA) and temperatures (273-298 K). The model was built in a systematic way with parameters that were, for an important part, calculated from independe nt experiments. With the constants used in the model not only the synthesis reaction but also phenylglycine amide hydrolysis and cephalexin hydrolysis could be described accurately. In contrast to the models described in lite rature, only a limited number (five) of constants was required to describe the reaction at a certain temperature. For the temperature dependency of th e constants, the Arrhenius equation was applied, with the constants at 293 K as references. Again, independent experiments were used, which resulted i n a model with high statistic reliability for the entire temperature range. Low temperatures were found beneficial for the process because more cephal exin and less phenylglycine is formed. The model was used to optimize the reaction conditions using criteria such as the yield on 7-ADCA or on activated phenylglycine. Depending on the weig ht of the criteria, either a high initial phenylglycine amide concentration (yield on 7-ADCA) or a high initial 7-ADCA concentration (yield on phenylg lycine amide) is beneficial. (C) 2001 John Wiley & Sons, Inc. Biotechnol Bi oeng 73: 171-178, 2001.